Glaucoma is the second leading cause of blindness overall in the United States and first among African Americans. Elevated intraocular pressure from defects in the iris and aqueous humor drainage system in the anterior segment frequently accompanies glaucoma. Retinal ganglion cell death leading to glaucoma apparently results from the insult of chronically high IOP but the underlying molecular mechanisms are not understood. The genetic regulatory networks governing normal development of anterior segment structures are also poorly understood. Mice provide ideal models for determining the basic mechanisms contributing to normal eye development and for molecularly analyzing genetic eye disease. Mutations in the bicoid-related homeobox gene PITX2 result in Axenfeld-Rieger Syndrome (ARS), an autosomal dominant disease resulting in congenital anterior segment defects and glaucoma. I have cloned Pitx2 from mice and used gene targeting to generate an allelic series (null, hypomorphic, and conditional) for Pitx2 in mice. Initial analysis of the null allele established a more widespread requirement for Pitx2 in eye development than predicted from the human phenotype. Homzygotes mutants have defects in the optic nerve and posterior parts of the eye. The goal of this proposal is to use these mice to analyze the basic mechanisms of Pitx2 function in normal eye development and to identify the molecular consequences of partial or complete loss of Pitx2 activity. We will assess the hypothesis that Pitx2 has distinct functions in the ocular neural crest and mesoderm lineages. Pitx2 expression in each lineage will be determined after using binary transgenic systems to mark each cell lineage. These marking systems will also be used to compare the fates of each lineage in wild type and Pitx2-/- eyes, and determine the genetic mechanisms that require Pitx2. ARS results from altered Pitx2 dosage, indicating certain steps in eye development are highly sensitive to varied PITX2 protein levels. We will vary Pitx2 gene dosage using the null and hypomorphic alleles to identify the specific developmental functions that are most to variations in PITX2 levels and determine the underlying molecular mechanisms. Finally, we will use chimeric mice to rescue the lethality of Pitx2-/- mice in order to analyze Pitx2 functions later in eye development. This multifaceted approach should provide specific mechanistic details about the multiple functions of Pitx2 in eye development and also promises to provide insight into more general fundamental mechanisms of periocular mesenchyme in development. This basic information is essential for understanding eye disease.